Studies over the last several years of mutations/polymorphisms in G-protein coupled receptor genes in the human population have revealed several classes of genetic variations. One class is composed of polymorphisms that alter receptor function or expression and are the direct cause of a disease. These diseases are typically rare and the polymorphism is not found in healthy (non-affected) individuals. Examples of this class are polymorphisms of the leutinizing hormone receptor that cause constitutive activation and results in familial male precocious puberty (Themmen, A. P. N. et al., J Endocrinol 153:179-183, 1997), the calcium sensing receptor which causes constitutive activation and results in familial hypoparathyroidism (Chattopadhyay, N. et al., Endocr Rev 17:289-307, 1996), and the V2 vasopressin receptor that causes depressed receptor function and results in nephrogenic diabetes insipidus (Rosenthal, W. A. et al., J. Biol. Chem. 268:13030-13033, 1993).
A second class of genetic variations of G-protein coupled receptors comprises those that alter receptor function or expression but do not appear to be the direct or sole cause of a disease. Here, the variation can be common in apparently healthy individuals. Studies to date indicate that these polymorphisms may act as disease modifiers (Liggett, S. B. 1996. The genetics of 2-adrenergic receptor polymorphisms: relevance to receptor function and asthmatic phenotypes. In The Genetics of Asthma. S. B. Liggett and D. A. Meyers, editors. Marcel Dekker, New York. 455478) with the physiologic consequences becoming apparent when receptor function is critical for compensation in the diseased state or for the response to therapy. As such, they may be responsible for certain clinical subsets of a given disease (such as different phenotypes of asthma or hypertension) or represent the basis of differential responsiveness to therapeutic agents.
β2-adrenergic receptors (β2AR) are G-protein coupled receptors that are activated by endogenous catecholamines. These receptors are widely distributed, and play important roles in regulating cardiac, vascular, pulmonary, and metabolic functions. Studies of such physiologic functions of β2AR in humans have revealed several observations. First, there appears to be substantial interindividual variation in responsiveness, and secondly receptor function appears to be dynamically regulated as indicated by intraindividual variation. Recently, significant genetic variability in the structure of the β2AR in the human population due to polymorphisms in the β2AR gene has been delineated (1, 2). These polymorphisms are located 46, 79, 100 and 491 bases downstream of the ATG start codon, and result in variation-that occurs in the amino-terminus of the receptor at amino acids 16 (Arg or Gly), 27 (Gln or Glu) and 34 (Val or Met) and in the fourth transmembrane spanning domain at amino acid 164 (Thr or Ile). In recombinant cell studies (3, 4), and in primary cultures of cells endogenously expressing these variants (5), clear phenotypic differences have been shown between the polymorphic receptors. The Gly16 receptor was found to undergo enhanced agonist-promoted downregulation of receptor number as compared to the Arg16 receptor (3). In contrast, the Glu27 receptor was found to undergo very little agonist-promoted downregulation compared to the Gln27 receptor (3). These variants are common in the population (1). The Ile164 receptor, which occurs in the heterozygous state in 5% of the population, displays depressed coupling to the stimulatory G protein, Gs (4).
Subsequent studies have assessed the role of the aforementioned polymorphic β2AR in diseases such as asthma [reviewed in (6)], based on the role of β2AR in modulating bronchial smooth muscle tone. In these studies, no differences in the frequencies of any of these polymorphisms between non-asthmatics and asthmatics have been reported. However, polymorphisms at positions 16 and 27 were found to act as significant disease modifiers (7-10). In the majority of the above cited studies, the presumption has been that the clinical phenotypes of those with the Gly16 polymorphism were due to enhanced downregulation of this receptor (as compared to those with the Arg16 receptor) by endogenous catecholamines. Thus responsiveness to β-agonists in individuals with this polymorphism has been considered depressed due to this tonic downregulation. A similar scenario is considered in individuals with the Glu27 variant, who exhibit greater responsiveness to β-agonists than those individuals with the Gln7 receptor, presumably due to its minimal downregulation by catecholamines. An amplification of these differences may occur during chronic agonist administration, as has recently been shown in asthma (11).
The β2AR is encoded by an intronless gene on chromosome 5q31 (12). Receptor transcripts have a 5′ leader region harboring an open reading frame (ORF) that encodes a 19 amino acid peptide (13). Recent in vivo and in vitro studies have shown that this peptide impedes translation of β2AR mRNA, and thus regulates cellular expression of the receptor (14). Given the importance of this 5′ leader cistron in controlling β2AR expression, this region in the human population was examined for genetic variability.
A Summary of the Invention
The present invention is based on the discovery of a common polymorphism that is located in the 5′ leader cistron (5′LC) of the β2AR gene. In particular, a polymorphism of cytosine or thymine located 47 bases upstream of the β2AR coding block results in either Arg or Cys being encoded at the terminal amino acid (position 19) of the 5′LC peptide (Arg19Cys) (see FIGS. 1-3; SEQ ID NOS:1-3, respectively). The inventor herein has also discovered that a substitution of Cys for Arg in the 5′LC peptide results in increased expression of β2AR in both recombinant and airway smooth muscle cells, which endogenously express β2AR. Thus, it is believed this β2AR 5′ LC polymorphic site represents, at least in part, the genetic basis of variable physiologic sympathetic responses, variation in disease phenotypes, and differences in the therapeutic efficacy of β-agonists and p-antagonists.
Thus, in one aspect the present invention provides methods and compositions for genotyping and haplotyping the β2AR gene of an individual. In one embodiment, a genotyping method comprises isolating from the individual a nucleic acid mixture comprising the two copies of the β2AR gene present in the individual and determining the identity of the nucleotide pair at the 5′LC polymorphic site in the two copies to assign a β2AR genotype to the individual. The haplotyping method comprises isolating from the individual a nucleic acid molecule containing only one of the two copies of the β2AR gene, or a fragment thereof, that is present in the individual and determining in that copy the identity of the nucleotide at the 5′LC PS and at one or more additional β2AR polymorphic sites. Compositions useful in performing the genotyping and haplotyping methods include oligonucleotide probes and primers designed to specifically hybridize to a target region containing the 5′LC polymorphic site. These genotyping methods and compositions are useful for studying the effect of the 5′LC polymorphisms in the etiology of various diseases and efficacy of drugs targeting the β2AR.
In another embodiment, the invention provides a method for detecting which variant(s) of the 5′LC peptide is expressed in an individual. The method comprises contacting a biological sample from the individual with a first antibody that specifically recognizes and binds to only one of the β2AR 5′LC peptide variants and detecting a complex formed with the first antibody. In a preferred embodiment, the method further comprises contacting the biological sample with a second antibody that specifically recognizes and binds to the other β2AR 5′LC peptide variant and detecting a complex formed with the second antibody. This method is useful for investigating the effects of the 5′LC peptide variants on β2AR expression. In addition, the preferred embodiment is useful to determine the genotype. If both antibodies react with the sample, the individual is heterozygous C/T at the 5′LC polymorphic site.
Another aspect of the invention is based on the discovery that β2AR alleles carrying the 5′LC C polymorphism are most likely to also have the polymorphisms which encode Gln and Arg at amino acids 16 and 27, respectively. Thus, the genotype for the 5′LC polymorphic site may be used to predict the identity of the genotype for one or both of these β2AR coding block polymorphisms.
In yet another embodiment, the invention provides a method for identifying an association between a β2AR 5′LC genotype and a trait. The method comprises comparing the frequency of the β2AR 5′LC genotype in a population exhibiting the trait with the frequency of the genotype in a reference population, wherein a higher frequency of the genotype in the trait population than in the reference population indicates the trait is associated with the genotype. Such methods have applicability in developing diagnostic tests and therapeutic treatments for a variety of diseases, including arrhythmia, heart failure, hypertension, vascular disease, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes and premature labor.
The present invention also provides a method for predicting an individual's genetic predisposition to a disease modified by the β2AR. The method comprises determining the individual's genotype for the 5′LC polymorphic site. If the individual is homozygous T, increased β2AR expression is likely and the individual has an increased risk for diseases affected by too much β2AR expression. If the individual is homozygous C, decreased β2AR expression is likely and the individual has an increased risk for diseases affected by too little β2AR expression. In a preferred embodiment, the disease modified by the β2AR is selected from the group consisting of arrhythmia, heart failure, hypertension, vascular disease, migraine, asthma, chronic obstructive pulmonary disease (COPD), anaphylaxis, obesity, diabetes and premature labor.
The present invention further provides methods for predicting a patient's response to β-agonist therapy for bronchospasm, which typically occurs in asthma, COPD and anaphylaxis. In one embodiment, the method comprises determining the genotype of the 5′LC polymorphic site in the patient's β2AR gene. If the patient is homozygous for the T polymorphism, failure to respond to a β-agonist is likely, while a patient is likely to respond if he or she is homozygous for the C polymorphism or is heterozygous TIC at this site. Thus, knowledge of a patient's β2AR 5′LC genotype provides a physician with information useful for making determinations as to which drug to administer, drug dosages, and duration of treatment.